In Vitro Modeling of and Automated Drug Screening in Embryonal Brain Tumors Using Tumor-Brain Organoids

Embryonal brain tumors (EBTs), arising during early brain development, present significant challenges in understanding pathogenesis and developing of treatments. This study explores the efficacy of forebrain organoid (FBO) models in replicating the complexities of the EBT microenvironment. We established an EBT-forebrain-organoid (TBO) model, incorporating embryonal tumor with multilayered rosettes (ETMR) and atypical teratoid and rhabdoid tumor (ATRT), using a scalable liquid-handling-assisted coaggregation method. Comprehensive characterization, including wholemount immunostaining, immunohisto-chemistry, single-cell RNA sequencing, integration with existing datasets, and automated cell type-specific drug screening, validated the TBO model. TBOs closely mimicked mid-gestational fetal brain environments, and EBTs in FBOs better approximated native intratumoral heterogeneity at both histological and transcriptomic levels than tumor-only 3D approaches. Drug screening in ETMR-FBOs identified promising treatment options, including anthracyclins and triptolide, demonstrating anti-tumoral effects with minimal neurotoxicity. Scalable TBO models that incorporate immature neural microenvironments better recapitulate individual tumor heterogeneity and mark significant progress towards targeted therapy and personalized precision medicine. Overall design: scRNA-seq (10xGenomics) of fresh tumor forbrain organoid samples (n = 2) and fixed snRNA-seq samples from tumors (n = 17) and cell lines (n = 10).